1. Field of the Invention
This invention relates to compositions and methods useful for measuring intracellular calcium, and, more specifically to novel fluorinated aromatic compounds.
2. Background
Calcium is a key element in the regulation of numerous cellular processes. Calcium is known to control the contraction of muscle, the secretion of hormones from gland cells and transmitters from nerve synapses, plus a multitude of other functions. For example, the development of tension in vascular smooth muscle and cardiac muscle is dependent on a rise in free cytosolic calcium ion (Ca.sup.++) levels.
Variations of the calcium ion concentration in the cytosol of cells exert profound effects on cellular metabolism, both in normal cell physiology, as well as in the mediation of the toxicito of various chemical and physical agents. For example, it is known that changes in levels of intracellular calcium ion are linked to physiological events as diverse as platelet aggregation, exocytosis and cell proliferation.
The role of calcium ion in important cellular processes has directed investigation into the development of techniques for measuring calcium ion levels in living cells. The use of optical indicators, in particular polycarboxylate chelating compounds, are among the most reliable methods of calcium ion detection currently available. These polycarboxylate compounds are introduced to the cell as ester derivatives which penetrate the cell membrane. Inside the cell, the esters are cleaved enzymatically to give polycarboxylate ions which are impermeable (or permeable only at a slow rate) to the cell membrane. Thus, once inside the cell, the polycarboxylate ions cannot escape at any appreciable rate. When the polycarboxylate compounds bind to calcium ion, a change in their spectral properties is produced. The magnitude of this spectral shift may be correlated to the local calcium ion concentration and thus be used to measure the amount of bound calcium ion. From a knowledge of this correlation, the intracellular calcium concentration can be determined.
Currently one popular embodiment of these methods includes monitoring the spectral properties of BAPTA (1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid) or analogs thereof, such as fura-2, indo-1, and fluo-2. These compounds can be easily loaded into cells by hydrolysis of their membrane-permeable esters (here, R is lower alkyl or acetoxymethyl). ##STR1##
Such conventional calcium ion indicators are designed to determine cytosolic calcium ion levels only. Unfortunately, the basal calcium ion levels in the cytosol and in various organelles within the cell can differ dramatically; hence, conventional indicators may not be suitable for carrying out measurements in organelles. For example, the calcium ion concentrations in the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) are thought to be several orders of magnitude greater than in the cytosol. Ionized calcium concentrations in the SR and the ER are of great interest. Calcium release from the SR is responsible for contraction in muscle, and the regulation of SR calcium uptake and release are of importance in muscle function. In addition, ER or related calcium sequestering organelles are important in receptor mediated calcium signalling in non-muscle cells. Yet present intracellular calcium ion indicators such as fura-2 are saturated at the calcium levels normally present in the SR and ER. Also, the use of high dissociation constant (K.sub.D) calcium indicators for cytosolic calcium ion measurements has several important advantages: (1) reduced buffering of intracellular calcium and reduced perturbation of calcium transients, and (2) measurement of more rapid calcium transients than with ordinary calcium chelators. Hence, a need for higher dissociation constant calcium ion indicators exists.
In addition, the role of calcium ions in maintaining normal physiological function and in the mediation of pathological conditions is barely understood. Nevertheless, it is already clear that there are significant perturbations in cytosolic calcium levels and transients which are associated with disease states. For example, studies in perfused rat heart have demonstrated that a significant increase in the level of cytosolic free calcium occurs prior to irreversible cell injury (Steenbergen et al., Circ. Res. 60, 700; 1987). Indeed, there is much literature suggesting that an elevation of cytosolic calcium ion concentration plays a general role in producing irreversible cell injury. Hence, it would be extremely useful if techniques and reagents were available which could be extended to the study of such pathological states. Such reagents would have to provide reliable measurements under conditions of high intracellular Ca.sup.+2 concentrations (i.e., have a high K.sub.D) and ideally have the ability to be introduced selectively into specific organelles. If appropriate and non-injurious loading conditions could be developed, these could also be used for the determination of free calcium ion concentrations in human subjects. This would be a major breakthrough for understanding the role of calcium ion in normal and pathological states, and potentially for the diagnosis of a bread range of diseases.
To date, conventional methods and agents have not proven to be effective for determining either the high levels of calcium ions associated with some pathological conditions or the calcium ion level in organelles of interest. For these reasons it would be desirable to provide improved calcium ion indicators and methods, which avoid the disadvantages of these conventional agents and methods, while providing effective means for determining calcium ion concentration.